Infection with HCV is a major cause of human liver disease throughout the world. In the U.S., an estimated 4.5 million Americans are chronically infected with HCV. Although only 30% of acute infections are symptomatic, greater than 85% of infected individuals develop chronic, persistent infection. Treatment costs for HCV infection have been estimated to be $5.46 billion for the US in 1997. Worldwide, over 200 million people are estimated to be chronically infected. HCV infection is responsible for 40-60% of all chronic liver disease and 30% of all liver transplants. Chronic HCV infection accounts for 30% of all cirrhosis, end-stage liver disease, and liver cancer in the U.S.
Due to the high degree of variability in the viral surface antigens, existence of multiple viral genotypes, and demonstrated specificity of immunity, the development of a successful vaccine in the near future is unlikely. Alpha-interferon (alone or in combination with ribavirin) has been widely used since its approval for treatment of chronic HCV infection. However, adverse side effects are commonly associated with this treatment: flu-like symptoms, leukopenia, thrombocytopenia, depression, as well as anemia induced by ribavirin (Lindsay, K. L. (1997) Hepatology 26 (suppl 1): 71S-77S). This therapy remains less effective against infections caused by HCV genotype 1 (which constitutes ˜75% of all HCV infections in the developed markets) compared to infections caused by the other 5 major HCV genotypes. Unfortunately, only ˜50-80% of the patients respond to this treatment (measured by a reduction in serum HCV RNA levels and normalization of liver enzymes) and, of responders, 50-70% relapse within 6 months of cessation of treatment. With the introduction of pegylated interferon (Peg-IFN), both initial and sustained response rates have improved substantially. However, the side effects associated with combination therapy and the impaired response in patients with genotype 1 present opportunities for improvement in the management of this disease.
First identified by molecular cloning in 1989 (Choo, Q-L et al (1989) Science 244:359-362), HCV is now widely accepted as the most common causative agent of post-transfusion non-A, non-B hepatitis (NANBH) (Kuo, G et al (1989) Science 244:362-364). Due to its genome structure and sequence homology, this virus was assigned as a new genus in the Flaviviridae family. Like the other members of the Flaviviridae, such as flaviviruses (e.g. yellow fever virus and Dengue virus types 1-4) and pestiviruses (e.g. bovine viral diarrhea virus, border disease virus, and classic swine fever virus) (Choo, Q-L et al (1989) Science 244:359-362; Miller, R. H. and R. H. Purcell (1990) Proc. Natl. Acad. Sci. USA 87:2057-2061), HCV is an enveloped virus containing a single strand RNA molecule of positive polarity. The HCV genome is approximately 9.6 kilobases (kb) with a long, highly conserved, noncapped 5′ nontranslated region (NTR) of approximately 340 bases which functions as an internal ribosome entry site (IRES) (Wang C Y et al ‘An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5’ noncoding region RNA—A Publication of the RNA Society. 1(5): 526-537, 1995 July). This element is followed by a region which encodes a single long open reading frame (ORF) encoding a polypeptide of ˜3000 amino acids comprising both the structural and nonstructural viral proteins.
Upon entry into the cytoplasm of the cell, this RNA is directly translated into a polypeptide of ˜3000 amino acids comprising both the structural and nonstructural viral proteins. This large polypeptide is subsequently processed into the individual structural and nonstructural proteins by a combination of host and virally-encoded proteinases (Rice, C. M. (1996) in B. N. Fields, D. M. Knipe and P. M. Howley (eds) Virology 2nd Edition, p 931-960; Raven Press, N.Y.). There are three structural proteins, C, E1 and E2. The P7 protein is of unknown function and is comprised of a highly variable sequence. There are several nonstructural proteins. NS2 is a zinc-dependent metalloproteinase that functions in conjunction with a portion of the NS3 protein. NS3 incorporates two catalytic functions (separate from its association with NS2): a serine protease at the N-terminal end, which requires NS4A as a cofactor, and an ATP-ase-dependent helicase function at the carboxyl terminus. NS4A is a tightly associated but non-covalent cofactor of the serine protease. NS5A is a membrane-anchored phosphoprotein that is observed in basally phosphorylated (56 kDa) and hyperphosphorylated (58 kDa) forms. While its function has not fully been elucidated, NS5A is believed to be important in viral replication. The NS5B protein (591 amino acids, 65 kDa) of HCV (Behrens, S. E. et at (1996) EMBO J. 151 2-22) encodes an RNA-dependent RNA polymerase (RdRp) activity and contains canonical motifs present in other RNA viral polymerases. The NS5B protein is fairly well conserved both intra-typically (˜95-98% amino acid (aa) identity across 1b isolates) and inter-typically (˜85% aa identity between genotype 1a and 1b isolates). The essentiality of the HCV NS5B RdRp activity for the generation of infectious progeny virions has been formally proven in chimpanzees (A. A. Kolykhalov et al. (2000) Journal of Virology, 74(4): 2046-2051). Thus, inhibition of NS5B RdRp activity (inhibition of RNA replication) is predicted to be useful to treat HCV infection.
Compounds useful for treating HCV-infected patients are desired which selectively inhibit HCV viral replication. In particular, compounds which are effective to inhibit the function of the NS5A protein are desired. The HCV NS5A protein is described, for example, in the following references: S. L. Tan, et al., Virology, 284:1-12 (2001); K.-J. Park, et al., J. Biol. Chem., 30711-30718 (2003); T. L. Tellinghuisen, et al., Nature, 435, 374 (2005); R. A. Love, et al., J. Virol, 83, 4395 (2009); N. Appel, et al., J. Biol. Chem., 281, 9833 (2006); L. Huang, J. Biol. Chem., 280, 36417 (2005); C. Rice, et al., WO2006093867; R. Hamatake, et al., Ann. Rep. Med. Chem. 47, 331 (2012); D. G. Cordek, et al., Drugs of the Future, 36, 691 (2011); U. Schmitz, et al., Recent Patents on Anti-Infective Drug Discovery, 3, 77 (2008).
Daclatasvir (BMS-790052) is the most advanced NS5A inhibitor in clinic development. It has demonstrated significant viral suppression in various phase 2 studies on treatment-naïve patients, especially with genotype 1b (GT 1b) infections. Clinical response of Daclatasvir for patients with GT 1a (GT 1a) infectious is often less profound. For example, in an exploratory study (A. Lok, et al., 61st AASLD, The Liver Meeting, Boston, Mass., Oct. 29-Nov. 2, 2010, LB-8) when eleven GT 1 patients of prior null responders were dosed with Daclatasvir and an NS3 protease inhibitor Asunaprevir, only four patients achieved the primary endpoint of SVR12 (sustained virological response 12 weeks after treatment). The primary reason for treatment failure was viral breakthrough which occurred in six patients infected with GT 1a HCV. In contrast, the two GT 1b-infected patients in this group both had SVR12. A recently published study has also confirmed that the impressive efficacy can be achieved against GT 1b (K. Chayama, et al., Hepatology, 55, 742 (2012)). This is consistent with the relative in vitro potency of Daclatasvir against GT 1b-NS5A-resistant variants and GT-1a NS5A-resistant variants. It has been demonstrated NS5A inhibitor-associated mutations arise predominantly at residue positions 28, 30, 31, and 93 in domain I of the NS5A protein (M. Gao, et al., Nature, 465, 96 (2010); R. A. Fridell, et al., Antimicrob. Agents Chemother. 54, 3641 (2010); R. E. Nettles, et al., Hepatolgy, 54, 1956 (2011)). The most prominent mutation affects Tyr93. Importantly, mutations at this site confer cross-resistance to several NS5A inhibitors and, in the case of daclatasvir, reduce drug sensitivity by ˜20-fold for genotype 1b subgenomic replicons and ˜1,800-fold for genotype 1a subgenomic replicons.
In another study (M. Sulkowski, et al., 47th EASL Congress, Barcelona, Spain, Apr. 18-22, 2012, P-1422) when GT 1, 2 or 3 treatment nave patients were dosed with Daclatasvir and a nucleotide NS5B inhibitor GS-7977. The treatment response rate was noticeably lower in GT 2 and 3 than GT1 cohorts. In a 3-day monotherapy proof-of-concept study when another NS5A inhibitor IDX719 was administered to GT 1-4 treatment naïve patients, HCV viral RNA reduction was found to be significantly lower in GT 2 cohorts than GT 1, 3 and 4 cohorts (www.idenix.com/hcv/IDX719_HCVClinPharmMtg_FINAL %206%2027%2012.pdf). These clinical findings are consistent with the in vitro data indicating that these drugs are less potent against certain HCV genotypes such as the GT 2a-J6 strain. For example, it has been reported that Daclatasvir was much less potent against GT 2a-J6 (EC50=7200 pM) than GT1a (EC50=8 pM) (M. Gao, et al., 46th EASL Congress, Berlin, Germany, Mar. 30-April 2011, P-787). The HCV GT 2a-J6 strain has a point substitution at L31M, which is clinically relevant to an estimated 80% of the GT 2a-infected patient population based on a sequence alignment analysis from an EU HCV sequence database euHCVdb (the European hepatitis C virus database, see also C. Combet, et al., Nucleic Acids Res. 2007, 35: D363-D366).
Based on the foregoing, there exists a significant need to identify compounds with the ability to inhibit HCV, especially compounds which are effective against GT 1a resistant variants and have activity against a broad range of HCV genotypes.